Agrobacterium, a natural plant pathogen, has been widely used for the transformation of dicotyledonous plants and more recently for transformation of monocotyledonous plants. The advantage of the Agrobacterium-mediated gene transfer system is that it offers the potential to regenerate transgenic cells at relatively high frequencies without a significant reduction in plant regeneration rates. Moreover, the process of DNA transfer to the plant genome is well characterized relative to other DNA delivery methods. DNA transferred via Agrobacterium is less likely to undergo any major rearrangements than is DNA transferred via direct delivery, and it integrates into the plant genome often in single or low copy numbers.
Currently the most commonly used Agrobacterium-mediated gene transfer system is a binary transformation vector system where the Agrobacterium has been engineered to include a disarmed, or nononcogenic, Ti helper plasmid, which encodes the vir functions necessary for DNA transfer, and a much smaller separate plasmid called the binary vector plasmid, which carries the transferred DNA, or the T-DNA region. The T-DNA is defined by sequences at each end, called T-DNA borders, which play an important role in the production of T-DNA and in the transfer process.
Agrobacterium-mediated introduction of multiple genes, or so-called co-transformation, occurs naturally. In nature, the Ti plasmid of both the octopine producing strain, A348, and the A. rhizogenes strains have multiple T-DNA regions that transfer independently and integrate into the plant genome.
Co-transformation has been carried out with genetically engineered Agrobacterium strains. Introduction of multiple genes into plants using Agrobacterium-mediated transformation systems may result in linked or unlinked integration of T-DNA regions depending upon the method of co-transformation used. In some instances, a transformed plant may have both linked and unlinked integration of the T-DNA regions when multiple T-DNA regions are transferred. As a result, subsequent generations of transformed plants may retain only subsets of the originally transferred and integrated T-DNA regions. Such segregation of T-DNA regions carrying different genes may be desirable when the T-DNA region or regions that are lost to segregating progeny carry selectable marker genes that are desirable for selecting initial transformed plants, but are undesirable in the progeny of those transformed plants.
Co-transformation in engineered Agrobacterium binary vector systems has thus far been accomplished in three different ways. The first of these methods involves coinfection of a plant with two Agrobacterium strains, each of which has a unique T-DNA carried on identical binary vector plasmids (McNight et al. (1987) Plant Mol. Biol. 8:439-445) or different binary vector plasmids (DeNeve et al. (1997) Plant J. 11(1):15-29. Alternatively, co-transformation may be achieved by infection of a plant with a single Agrobacterium strain that has one T-DNA residing on the Ti helper plasmid and a second T-DNA residing on a binary plasmid (de Framond et al. (1986) Mol. Gen. Genet. 202:125-131). A third method of co-transformation involves infection of a plant with a single Agrobacterium strain having two separate T-DNA regions on a single binary plasmid (Komari et al. (1996) Plant J. 10(1):165-174). The first two methods favor unlinked integration of T-DNA regions, while the latter method favors linked integration of the T-DNA regions, though both types of integration may occur within one plant.
Given the advantages of Agrobacterium-mediated transformation systems and the need for simultaneous transformation of plants with more than one gene of interest, additional methods for efficient co-transformation using Agrobacterium are needed.